RADIATION INACTIVATION STUDIES OF THE DOPAMINE REUPTAKE TRANSPORTER PROTEIN

Using radiation inactivation, we have estimated the target size for th e neuronal dopamine transporter protein. The specific binding of sever al radioligands previously shown to label the dopamine transporter was determined in an irradiated striatal membrane preparation. The appare nt target size of the 1-[1-(2-[H-3] benzo[b]thienyl)cyclohexyl] piperi dine site was approximately 98 kDa. However, the apparent target size of the ''cocaine binding site,'' as measured with the cocaine analogue 2 beta-[H-3]carbomethoxy-3 beta-(4-fluorophenyl)tropane in the same a ssays, was approximately 140 kDa. Radiation inactivation of the bindin g of other ligands (GBR-12935 and mazindol) led to target size estimat es in the same range (94 kDa and 133 kDa, respectively). All of these target sizes are significantly larger than the estimate of 70 kDa deri ved from the deduced amino acid sequence for the cloned dopamine reupt ake transporter cDNA. Larger target sizes than expected have also been reported for ligand binding to the sodium-dependent serotonin transpo rter and glucose transporter. The estimated sizes for the ligand bindi ng site(s) associated with the dopamine transporter protein are diffic ult to reconcile with a single transporter protein of 70 kDa. We concl ude that the dopamine transporter protein is a homo- or hetero-oligome r when occupied in situ by uptake-blocking drugs like cocaine.